Cell growth, which is a process involving proliferation and division of cells, occurs in response to various stimuli.
Pathological conditions caused by hyperproliferation of cells, such as cancer, are characterized by uncontrollable cell cycle progression and thus excessive progression of the cell cycle, for example, resulting from abnormality in genes or proteins that directly or indirectly regulate the cell cycle progression. Substances that regulate hyperproliferation of cells through control of the cell cycle can be used for the treatment of various pathological conditions characterized by uncontrollable or unwanted cell growth.
Cell cycle progression is a complicated process involving highly regulated transition of phases and multiple checkpoints.
Cyclin-dependent kinases and associated serine/threonine protein kinases are important intracellular enzymes that play essential roles in the regulation of division and proliferation of cells. Catalytic subunits of cyclin-dependent kinases are activated by regulatory subunits known as cyclins, and multiple cyclins have been identified in mammals (NPL 1).
The retinoblastoma (Rb) protein is a checkpoint protein for transition from the G1 phase to the S phase in the cell cycle. The Rb protein associates with the E2F transcription factor family and inhibits the activity thereof in the absence of appropriate growth stimulation (NPLs 2 and 3). A cell stimulated by a mitogen enters the S phase through synthesis of cyclin D, which is a CDK4/6 activator. The cyclin D-bound CDK 4/6 inactivates the Rb protein through phosphorylation. The phosphorylation of the Rb protein releases E2F in order to indirective the transcription of a gene necessary for the S phase. The complete inactivation of the Rb protein requires phosphorylation of both cyclin D-CDK4/6 and cyclin E-CDK2. The phosphorylation of the Rb protein by CDK4/6 at a specific site is essential in the phosphorylation of cyclin E-CDK2 (NPL 4). Thus, cyclin D-CDK4/6 is an important enzyme complex which controls the transition from the G1 phase to the S phase.
CDK2 forms a complex with cyclin E and also forms a complex with cyclin A. CDK2 also acts on steps subsequent to the S phase and is responsible for DNA replication. The inhibition of CDK2 probably leads to the expression of genotoxicity (NPL 5).
Cyclin D has a molecular mechanism that positively regulates the activity of CDK4/6. In contrast, p16 encoded by the INK4a gene negatively regulates the activity of CDK4/6 (NPL 6).
CDK inhibitors can be used for the treatment of various diseases caused by abnormal cell growth, such as cancer, cardiovascular disorder, renal disease, specific infections, and autoimmune diseases. CDK inhibitors is also expected to be effective for the treatment of diseases including but not limited to rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, and cancer. The inhibition of cell cycle progression and cell growth through CDK inhibition is expected to be effective for such a disease on the basis of the technical findings described below.
Rheumatoid arthritis involves the formation of pannus through hyperproliferation of synovial cells. This hyperproliferation can be reduced by the introduction of p16 into an affected area of a model animal or the administration of a CDK4/6 inhibitor to the animal (NPLs 7 to 9). A CDK4-cyclin D complex regulates the production of MMP3 in synovial cells derived from a patient with rheumatoid arthritis. The negative regulation of the activity of CDK4/6 inhibits not only the proliferation but also production of MMP3 (NPL 10).
Thus, CDK4/6 inhibitors are expected to exhibit both an inhibitory effect on proliferation of synovial cells and a cartilage protective effect in rheumatoid arthritis.
A pathway for the regulation of cell growth including genes responsible for the checkpoints in the G1 and S phases of the cell cycle is associated with plaque progression, stenosis, and restenosis after angiogenesis. The overexpression of the CDK inhibitory protein p21 inhibits angiogenesis and subsequent growth of vascular smooth muscle and intimal hyperplasia (NPLs 11 and 12).
Abnormal regulation of the cell cycle is also associated with polycystic kidney disease, which is characterized by growth of cysts filled with fluid in the renal tubule. A small-molecule CDK inhibitor is effective for the treatment of the disease (NPL 13).
The induction of expression of the cell cycle inhibitory protein p21 with an adenoviral vector is effective in a murine pulmonary fibrosis model (NPL 14).
The level of cyclin D1/CDK4 is known to increase in a rat cerebral infarction model in association with neuronal death caused by local ischemia. The neuronal death is reduced by administering flavopiridol, which is a nonselective CDK inhibitor (NPL 15).
The cyclin D-CDK4/6-INK4a-Rb pathway is frequently detected in human cancer caused by abnormality of any factors contributing to growth of cancer cells, such as loss of functional p16INK4a, overexpression of cyclin D1, overexpression of CDK4, or loss of functional Rb (NPLs 16 to 18). Such abnormality promotes the cell cycle progression from the G1 phase to the S phase, and this pathway certainly plays an important role in oncogenic transformation or abnormal growth of cancer cells.
CDK4/6 inhibitors may be effective, particularly for tumors involving abnormality in genes that activate the CDK4/6 kinase activity, such as cancers involving the translocation of cyclin D, cancers involving the amplification of cyclin D, cancers involving the amplification or overexpression of CDK4 or CDK6, and cancers involving the inactivation of p16. CDK4/6 inhibitors may be effective for the treatment of cancers involving genetic abnormality in the upstream regulator of cyclin D, the amount of which increases due to defects in the upstream regulator.
In fact, many compounds that inhibit the CDK4/6 activity have been synthesized and disclosed in the art, and such compounds have been clinically tested for the treatment of cancers, such as breast cancer (NPL 19).
Most acute and severe radiotherapeutic and chemotherapeutic toxicities are caused by the effects on stem cells and progenitor cells. A CDK4/6 inhibitor causes temporary cell cycle arrest to hematopoietic stem and progenitor cells, and protects them from radiotherapeutic or chemotherapeutic cytotoxicity. After the treatment with the inhibitor, hematopoietic stem and progenitor cells (HSPCs) return from the temporary dormancy and then function normally. Thus, the chemotherapeutic resistance with use of a CDK4/6 inhibitor is expected to provide a significant protection of bone marrow (NPL 20).
Hence, CDK4/6 inhibitors are expected to be effective for the treatment of rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, or cancer, and the protection of bone marrow, in particular, for the treatment of rheumatoid arthritis or cancer and the protection of bone marrow.
PTL 1 and NPL 21 disclose CDK4 inhibitors, PTLs 2 and 3 and NPLs 22 to 24 disclose CDK4/6-containing CDK inhibitors, and NPL 25 discloses CDK4/FLT3 inhibitors.
Pyrido[3,4-d]pyrimidine derivatives exhibit an inhibitory effect on Mps1 (also known as TTK) (PTL 4). This inhibitory effect is completely different from the CDK4/6 inhibitory effect disclosed in the present invention.
NPL 26 and NPL 27 disclose that a plurality of pyrido[3,4-d]pyrimidine derivatives exhibit a CDK2 inhibitory activity, which is completely different from the superior CDK4/6 inhibitory effect exhibited by the present invention.